Discovery of new transcription factors in Pyrococcus furiosus with DNA-affinity protein capture and mass spectrometry
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Gene expression is often under regulation at the transcriptional level, controlled by transcription factors. The primary goal of this research project is to discover and characterize new transcription factors in Pyrococcus furiosus (Pf), a model archaeal organism. The transcription system of archaea has both bacterial-like and eukaryotic-like features. Only a very limited number of archaeal transcription regulatory proteins have been identified to date, and transcription regulatory pathways are far from being well known. In this project transcription factors were discovered using DNA affinity protein capture followed by mass spectrometry based protein identification. First the method was tested by the protein capture and identification of LrpA, a known transcription factor. The native LrpA from Pf cell extract was successfully identified using the optimized protocol. Next, the Pf gene expression profile on the genome-wide scale has been described and compared between two different cell-growth conditions with maltose or peptides as the primary carbon source. Additional bioinformatics analysis identified a conserved palindromic sequence in the putative promoter region of several genes that are up-regulated by growth on maltose and all are involved in glycolysis. Based on these analyses, the DNA fragments upstream of the open reading frame of PF0272 and PF0464, which encode 4-±-glucanotransferase and glyceraldehyde-3-phosphate ferredoxin oxidoreductase, were selected for DNA-affinity protein capture. A novel transcription regulatory protein PF0124 (PF0124p) was identified and its specific DNA binding ability at sites upstream of multiple genes was confirmed by the electrophoretic mobility shift assay. DNA footprinting showed that PF0124p binds to a region upstream of both PF0272 and PF0464, which contains the conserved palindromic motif. Furthermore, PF0124p was shown to repress both PF0272 and PF0464 gene transcription in cell-free transcription assays. These results suggest PF0124p is a transcriptional regulator recognizing a conserved palindromic motif and possibly regulating a group of genes in the glycolysis pathway in Pyrococcus furiosus.